ABSTRACT The innate immune response, particularly the interferon (IFN) response, is the first line of defense against viral infections. This response includes upregulation of hundreds of IFN stimulate genes (ISGs), some of which have antiviral properties. Several ISGs are known to inhibit HIV in cell culture models, but there is little data on whether these or other ISGs are active against HIV in biologically relevant systems or indeed whether they are even present in HIV infected individuals and thus capable of impacting HIV replication in that context. This is because it is unknown which ISGs are induced by HIV infection and thus likely to contribute to the early antiviral response to HIV. We propose to address these gaps in this key area of HIV pathogenesis research by defining the ISGs that are present in early HIV infection and by comprehensively defining the ISGs that can inhibit HIV in the major target cell of infection, CD4+ T lymphocytes. We will examine the early IFN signature to HIV, both the ISGs that dominate overall - and thus may represent the major challenge to the establishment of HIV persistence - as well as the specific ISGs expressed by key innate effector cells such as NK cells. This will provide the first detailed view of the IFN antiviral response in humans exposed to HIV. A particularly critical aspect that enables this innovative work is the Mombasa Cohort, a long-term open cohort study of sex workers at risk for HIV that is the longest standing cohort of high-risk women globally. We will pair this with testing the specific antiviral properties of ISGs identified in this setting as well as those identified in a CRISPR/Cas9 inactivation screen in primary T lymphocytes to define their effects on HIV replication. We will do this using biologically relevant cells and transmitted viruses so that our studies are optimized to understand HIV transmission in humans. Studies in primary T cells with viruses that are spreading to humans will advance our current knowledge in this area, which has long focused on the inhibitory effects of ISGs in cell lines with lab-adapted viruses because of the challenges of human studies and studies in biologically relevant culture systems. We are well poised for this work because of the considerable optimization we have done to make large scale CRISPR/Cas9 inactivation screens in primary T lymphocytes feasible, our newly developed methods to study the NK antiviral response to HIV infection and our experience with RNA-seq methods. This proposal is novel in drilling down to define which of the ISGs present in early HIV infection are functionally important for restricting HIV replication. Defining these host antiviral effectors will provide mechanistic insights that may in turn open avenues to harness the virus/host arms race in favor of the host.